Prior to the present invention, as shown by Uzgiris et al., Two-Dimensional Crystallization Technique for Imaging Macromolecules with Application to Antigen-Antibody-Complement Complexes, Nature, Vol. 301, January 1983, pp. 125-129, techniques were available for growing protein arrays on lipid monolayers. Although the procedures described by Uzgiris et al. resulted in the production of ordered arrays of molecules as determined by optical defraction and by image processing of electron micrographs, the resulting ordered arrays were of small size and comprised a low portion of the total surface area as observed under an electron microscope. New techniques are therefore constantly being sought to improve the procedure for making ordered protein monolayers suitable for high resolution studies with electron microscopy as well as applications requiring ordered macromolecular films.
Experience has shown that conditions favoring two-dimensional crystallization of ordered monolayers require (1) fixation of molecules on a plane, (2) mobility of the molecules within the plane to allow sampling of various bonding arrangements, (3) identical orientation of all the molecules and (4) high concentration of molecules in the plane so that crystallization will be favored over two-dimensional liquid-like disorder. Depending upon the crystal nucleation rate for the bound protein, the surface of the lipid monolayer can quickly become saturated with bound protein yielding a highly dense and disordered condition referred to sometimes as the "protein jamming limit". It has been observed that once the jamming limit has been exceeded on the phospholipid monolayer, two-dimensional crystal growth rarely occurs.
The present invention is based on the discovery that significant improvements in crystallization of various proteins can be achieved by allowing the nucleation of the protein to occur on a phospholipid substrate by a slow diffusion limited process in a saline solution of the protein. Another procedure is allowing a monolayer of the protein to grow on the surface of a phospholipid film by conventional techniques as previously described above in Uzgiris et al. in Nature and thereafter transferring such coated phospholipid film to a different solution to promote the nucleation of the initial protein monolayer in the presence of the same protein at a lower concentration. A third procedure is transferring the coated phospholipid film to another solution having a different protein, or a salt solution, at a concentration different from the concentration of the protein in the original solution. In instances where a salt solution is used, a range of 0.15M to 0.6M is preferred particularly for promoting antibody crystal growth, and divalent ions such as calcium and magnesium have been found effective.